Program Director/Principal Investigator (Last, First, Middle): Escalante, Jorge C. PROJECT SUMMARY/ABSTRACT This MIRA proposal brings together two fields of prokaryotic metabolism and physiology that the PI?s group has contributed extensively to. The first is the assembly of the structurally complex coenzyme B12 (CoB12), and the second one is the regulation of protein function by lysine acetylation in response to metabolic stress. Previous work by the PI?s group in these areas has resulted in the discovery of new enzymes and pathways, and has established fundamental physiologic paradigms that apply to cells of all domains of life. We have learned a great deal about how the complex coenzyme B12 is made, and yet, gaps in our knowledge about its assembly remain. Although the remaining gaps are challenging to solve, recent breakthroughs in our group have generated the tools to address these questions and advance our understanding of the physiological integration of CoB12 biosynthesis in microorganisms of societal importance. We will investigate how the lower ligand base of CoB12 is synthesized and activated to its riboside in human pathogens, how vitamin B12 is converted to CoB12 in several Gram-positive pathogens, and why the last steps of the pathway occur at the cell membrane in all CoB12 producers known to date. Most of the proposed work will be performed in Salmonella enterica because of our deep knowledge of CoB12 biosynthesis in this bacterium, and the sophisticated genetic system available to do in vivo work. We will also use Salmonella to establish the function of heterologous, putative CoB12 biosynthetic functions in other bacteria and archaea. We will continue to investigate the role of lysine acetylation in the control of metabolic stress. Lysine acetylation is a posttranslational modification of profound relevance to human health and biotechnology. The impact of this regulatory mechanism on human cell aging and cancer, neurodegenerative diseases, diabetes, obesity, antimicrobial resistance, microbial pathogenesis, and other research areas of societal relevance emphasizes the need to continue advancing this field of research. Fundamental questions about lysine acetylation remain unanswered. The proposed work will investigate new role(s) of prokaryotic sirtuin deacetylases in prokaryotic physiology, and will continue to elucidate the functions and physiological roles of acetyltransferases in Gram-negative and Gram-positive human pathogens. Our findings obtained from experiments performed with prokaryotic model organisms will inform how the system may work in higher forms of life. A powerful, innovative combination of approaches, including transition metal spectroscopy, structural biology (crystallography), biochemistry, molecular biology, in vivo genetics, physiology, single-molecule biophysics, and system-wide analyses will be applied during the performance of the proposed work. We will collaborate with experts in the fields of spectroscopy, crystallography, molecular biophysics, and membrane biology to provide comprehensive, rigorous testing of hypotheses and working models. OMB No. 0925-0001/0002 (Rev. 03/16 Approved Through 10/31/2018) Page Continuation Format Page